Total bacterial count and somatic cell count in bulk and individual goat milk around kidding: Two longitudinal observational studies.

Total bacterial count (TBC) and SCC are important quality parameters in goat milk. Exceeding the bulk milk TBC (BMTBC) thresholds leads to price penalties for Dutch dairy goat farmers. Controlling these milk quality parameters can be challenging, especially around kidding. First, we describe the variation and the peaks around kidding of TBC and SCC in census data on Dutch bulk milk over the last 22 yr. Second, to explore causes of these elevations, we studied the variation of TBC and SCC in individual goat milk from 3 wk before to 5 wk after kidding and their association with systemic response markers IFN-γ, calprotectin, BHB, BCS, and fecal consistency. We visited 4 Dutch dairy goat farms weekly for 10 to 16 wk around kidding. Some of the goats had been dried off; other goats were milked continuously throughout pregnancy. A total of 1,886 milk samples from 141 goats were collected for automated flow cytometric quantification of TBC and SCC measurement. IFN-γ, calprotectin, and BHB were determined twice in blood of the same goats; most samples were collected after kidding. The BCS and fecal consistency were scored visually before and after kidding. We found a strong correlation between TBC and SCC (Spearman's rho = 0.87) around kidding. Furthermore, in the third week before kidding, the average TBC (5.67 log10 cfu/mL) and SCC (6.70 log10 cells/mL) were significantly higher compared with the fifth week after kidding, where the average TBC decreased to 4.20 log10 cfu/mL, and the average SCC decreased to 5.92 log10 cells/mL. In multivariable linear regression models, farm and stage of lactation were significantly associated with TBC and SCC, but none of the systemic response markers correlated with TBC or SCC. In conclusion, TBC and SCC in dairy goats were high in late lactation and decreased shortly after parturition. For SCC, the dilution effect might have caused the decrease, but this was not plausible for TBC. Moreover, the excretion of bacteria and cells in goat milk was not associated with the selected systemic response markers that were chosen as a readout for general immunity status, intestinal health, and metabolic diseases. Therefore, we assume that the TBC increase before kidding and the decrease after parturition are caused by other systemic, possibly hormonal, processes. To reduce BMTBC and bulk milk SCC, it would be advisable to keep milk of goats with highest numbers of bacteria and cells in their milk out of the bulk milk during end lactation. Further studies are needed to investigate the effects of withholding this end-lactation milk from the bulk tank.

Host genotype affects endotoxin release in excreta of broilers at slaughter age.

Host genotype, early post-hatch feeding, and pre- and probiotics are factors known to modulate the gut microbiome. However, there is a knowledge gap on the effect of both chicken genotype and these dietary strategies and their interplay on fecal microbiome composition and diversity, which, in turn, can affect the release of endotoxins in the excreta of broilers. Endotoxins are a major concern as they can be harmful to both animal and human health. The main goal of the current study was to investigate whether it was possible to modulate the fecal microbiome, thereby reducing endotoxin concentrations in the excreta of broiler chickens. An experiment was carried out with a 2 × 2 × 2 factorial arrangement including the following three factors: 1) genetic strain (fast-growing Ross 308 vs. slower growing Hubbard JA757); 2) no vs. combined use of probiotics and prebiotics in the diet and drinking water; and 3) early feeding at the hatchery vs. non-early feeding. A total of 624 Ross 308 and 624 Hubbard JA757 day-old male broiler chickens were included until d 37 and d 51 of age, respectively. Broilers (N = 26 chicks/pen) were housed in a total of 48 pens, and there were six replicate pens/treatment groups. Pooled cloacal swabs (N = 10 chickens/pen) for microbiome and endotoxin analyses were collected at a target body weight (BW) of 200 g, 1 kg, and 2.5 kg. Endotoxin concentration significantly increased with age (p = 0.01). At a target BW of 2.5 kg, Ross 308 chickens produced a considerably higher amount of endotoxins (Δ = 552.5 EU/mL) than the Hubbard JA757 chickens (p < 0.01). A significant difference in the Shannon index was observed for the interaction between the use of prebiotics and probiotics, and host genotype (p = 0.02), where Ross 308 chickens with pre-/probiotics had lower diversity than Hubbard JA757 chickens with pre-/probiotics. Early feeding did not affect both the fecal microbiome and endotoxin release. Overall, the results suggest that the chicken genetic strain may be an important factor to take into account regarding fecal endotoxin release, although this needs to be further investigated under commercial conditions.

Genetic relationship between poultry and wild bird viruses during the highly pathogenic avian influenza H5N6 epidemic in the Netherlands, 2017-2018.

In the Netherlands, three commercial poultry farms and two hobby holdings were infected with highly pathogenic avian influenza (HPAI) H5N6 virus in the winter of 2017-2018. This H5N6 virus is a reassortant of HPAI H5N8 clade 2.3.4.4 group B viruses detected in Eurasia in 2016. H5N6 viruses were also detected in several dead wild birds during the winter. However, wild bird mortality was limited compared to the caused by the H5N8 group B virus in 2016-2017. H5N6 virus was not detected in wild birds after March, but in late summer infected wild birds were found again. In this study, the complete genome sequences of poultry and wild bird viruses were determined to study their genetic relationship. Genetic analysis showed that the outbreaks in poultry were not the result of farm-to-farm transmissions, but rather resulted from separate introductions from wild birds. Wild birds infected with viruses related to the first outbreak in poultry were found at short distances from the farm, within a short time frame. However, no wild bird viruses related to outbreaks 2 and 3 were detected. The H5N6 virus isolated in summer shares a common ancestor with the virus detected in outbreak 1. This suggests long-term circulation of H5N6 virus in the local wild bird population. In addition, the pathogenicity of H5N6 virus in ducks was determined, and compared to that of H5N8 viruses detected in 2014 and 2016. A similar high pathogenicity was measured for H5N6 and H5N8 group B viruses, suggesting that biological or ecological factors in the wild bird population may have affected the mortality rates during the H5N6 epidemic. These observations suggest different infection dynamics for the H5N6 and H5N8 group B viruses in the wild bird population.

Molecular analysis of three Clostridium difficile strain genomes isolated from pig farm-related samples.

Clostridium difficile is an anaerobic spore-forming bacillus that usually causes gastrointestinal disorders in man and other animal species. Most of the strains isolated from animals are toxigenic being the virulent ribotype (RT) 078 predominant in several animal species. Although C. difficile is pathogenic to both humans and animals, there is no direct evidence of zoonosis. Deep genome sequencing provides sufficient resolution to analyse which strains found in animals might be related to human pathogens. So far, there are only a few fully sequenced genomes of C. difficile strains isolated from domestic and wild animals. Using Illumina technology, we have sequenced the genome of three isolates; a strain isolated from the vagina of a sow (5754), one from rat (Rattus spp) intestinal content (RC10) and a third one isolated from environmental rat faeces (RF17). Both, rat and rat faeces were sampled in fattening pig farms. Our study reveals a close genetic relationship of two of these isolates with the virulent strain M120 (RT078) isolated from a human patient. The analysis of the sequences has revealed the presence of antibiotic resistance genes, mobile elements, including the transposon linked with virulence Tn6164, and the similarity of virulence factors between these isolates and human strains. This is the first study focused on the sequencing of C. difficile genomes obtained from wild animals like rats, which can be considered as potential reservoirs for humans and other animal species. This study can help to understand the genome composition and epidemiology of this bacterium species.

Protecting effect of PrP codons M142 and K222 in goats orally challenged with bovine spongiform encephalopathy prions.

Breeding towards genetic resistance to prion disease is effective in eliminating scrapie. In sheep, classical forms of scrapie have been eradicated almost completely in several countries by breeding programs using a prion protein (PrP) gene (PRNP) amino acid polymorphism. For goats, field and experimental studies have provided evidence for several amino acid polymorphisms that are associated with resistance to scrapie, but only limited data are available concerning the susceptibility of caprine PRNP genotypes to BSE. In this study, goat kids representing five PRNP genotypes based on three polymorphisms (M142, Q211 and K222 and the wild type I142, R211 and Q222) were orally challenged with bovine or goat BSE. Wild type goats were killed with clinical signs between 24-28 months post inoculation (mpi) to both challenges, and goats with genotype R/Q211 succumbed between 29-36 mpi. I/M142 goats developed clinical signs at 44-45 mpi and M/M142 goats remained healthy until euthanasia at 48 mpi. None of the Q/K222 goats showed definite clinical signs. Taken together the highest attack ratios were seen in wild type and R/Q211 goats, and the lowest in I/M142, M/M142 and Q/K222. In all genotype groups, one or more goats remained healthy within the incubation period in both challenges and without detectable PrP deposition in the tissues. Our data show that both the K222 and M142 polymorphisms lengthen the incubation period significantly compared to wild type animals, but only K222 was associated with a significant increase in resistance to BSE infection after oral exposure to both BSE sources.

Variation in the prion protein sequence in Dutch goat breeds.

Scrapie is a neurodegenerative disease occurring in goats and sheep. Several haplotypes of the prion protein increase resistance to scrapie infection and may be used in selective breeding to help eradicate scrapie. In this study, frequencies of the allelic variants of the PrP gene are determined for six goat breeds in the Netherlands. Overall frequencies in Dutch goats were determined from 768 brain tissue samples in 2005, 766 in 2008 and 300 in 2012, derived from random sampling for the national scrapie surveillance without knowledge of the breed. Breed specific frequencies were determined in the winter 2013/2014 by sampling 300 breeding animals from the main breeders of the different breeds. Detailed analysis of the scrapie-resistant K222 haplotype was carried out in 2014 for 220 Dutch Toggenburger goats and in 2015 for 942 goats from the Saanen derived White Goat breed. Nine haplotypes were identified in the Dutch breeds. Frequencies for non-wild type haplotypes were generally low. Exception was the K222 haplotype in the Dutch Toggenburger (29%) and the S146 haplotype in the Nubian and Boer breeds (respectively 7 and 31%). The frequency of the K222 haplotype in the Toggenburger was higher than for any other breed reported in literature, while for the White Goat breed it was with 3.1% similar to frequencies of other Saanen or Saanen derived breeds. Further evidence was found for the existence of two M142 haplotypes, M142 /S240 and M142 /P240 . Breeds vary in haplotype frequencies but frequencies of resistant genotypes are generally low and consequently selective breeding for scrapie resistance can only be slow but will benefit from animals identified in this study. The unexpectedly high frequency of the K222 haplotype in the Dutch Toggenburger underlines the need for conservation of rare breeds in order to conserve genetic diversity rare or absent in other breeds.

Prion Type-Dependent Deposition of PRNP Allelic Products in Heterozygous Sheep.

UNLABELLED: Susceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild-type homozygous ARQ/ARQ (where each triplet represents the amino acids at codons 136, 154, and 171, respectively) sheep, scrapie susceptibility is reduced to near resistance in ARR/ARR animals while it is strongly enhanced in VRQ/VRQ carriers. Heterozygous ARR/VRQ animals exhibit delayed incubation periods. In bovine spongiform encephalopathy (BSE) infection, the polymorphism effect is quite different although the ARR allotype remains the least susceptible. In this study, PrP allotype composition in protease-resistant prion protein (PrP(res)) from brain of heterozygous ARR/VRQ scrapie-infected sheep was compared with that of BSE-infected sheep with a similar genotype. A triplex Western blotting technique was used to estimate the two allotype PrP fractions in PrP(res) material from BSE-infected ARR/VRQ sheep. PrP(res) in BSE contained equimolar amounts of VRQ- and ARR-PrP, which contrasts with the excess (>95%) VRQ-PrP fraction found in PrP in scrapie. This is evidence that transmissible spongiform encephalopathy (TSE) agent properties alone, perhaps structural aspects of prions (such as PrP amino acid sequence variants and PrP conformational state), determine the polymorphic dependence of the PrP(res) accumulation process in prion formation as well as the disease-associated phenotypic expressions in the host. IMPORTANCE: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative and transmissible diseases caused by prions. Amino acid sequence variants of the prion protein (PrP) determine transmissibility in the hosts, as has been shown for classical scrapie in sheep. Each individual produces a separate PrP molecule from its two PrP gene copies. Heterozygous scrapie-infected sheep that produce two PrP variants associated with opposite scrapie susceptibilities (136V-PrP variant, high; 171R-PrP variant, very low) contain in their prion material over 95% of the 136V PrP variant. However, when these sheep are infected with prions from cattle (bovine spongiform encephalopathy [BSE]), both PrP variants occur in equal ratios. This shows that the infecting prion type determines the accumulating PrP variant ratio in the heterozygous host. While the host's PrP is considered a determining factor, these results emphasize that prion structure plays a role during host infection and that PrP variant involvement in prions of heterozygous carriers is a critical field for understanding prion formation.

Phylogenetic analysis of highly pathogenic avian influenza A(H5N8) virus outbreak strains provides evidence for four separate introductions and one between-poultry farm transmission in the Netherlands, November 2014.

Phylogenetic analysis of highly pathogenic avian influenza A(H5N8) virus strains causing outbreaks in Dutch poultry farms in 2014 provides evidence for separate introduction of the virus in four outbreaks in farms located 16-112 km from each other and for between-farm transmission between the third and fourth outbreak in farms located 550 m from each other. In addition, the analysis showed that all European and two Japanese H5N8 virus strains are very closely related and seem to originate from a calculated common ancestor, which arose between July and September 2014. Our findings suggest that the Dutch outbreak virus strain 'Ter Aar' and the first German outbreak strain from 2014 shared a common ancestor. In addition, the data indicate that the Dutch outbreak viruses descended from an H5N8 virus that circulated around 2009 in Asia, possibly China, and subsequently spread to South Korea and Japan and finally also to Europe. Evolution of the virus seemed to follow a parallel track in Japan and Europe, which supports the hypothesis that H5N8 virus was exchanged between migratory wild waterfowl at their breeding grounds in Siberia and from there was carried by migrating waterfowl to Europe.

Q fever diagnosis and control in domestic ruminants.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii, a highly infectious agent that can survive in the environment. Therefore, Q fever has a major public health impact when outbreaks occur. Small ruminants are identified as the source in the majority of outbreaks in humans. Accurate diagnosis and effective control strategies are necessary to limit the zoonotic and veterinary impact of Q fever. For this, knowledge of the pathogenesis of Q fever and excretion routes of C. burnetii from infected animals is crucial. Abortions as well as normal parturitions in infected small ruminants are the most important excretion routes of C. burnetii. Excretion of C. burnetii via faeces and vaginal mucus has also been suggested. However, contamination of these samples by bacteria present in the environment may influence the results. This hampers the accurate identification of infected animals by these samples; however, the detection of C. burnetii in milk samples seems not to be influenced by environmental contamination. Q fever in animals can be detected by direct (immunohistochemistry and PCR) and indirect (complement fixation test (CFT), enzyme- linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) methods. A combination of both direct and indirect methods is recommended in current protocols to detect Q fever on herd level. For the control of Q fever in domestic animals, vaccination with a phase 1 C. burnetii whole cell inactivated vaccine is reported to be effective in preventing abortion and reducing bacterial shedding, especially after several years of administration. Vaccination might not be effective in already infected animals nor in pregnant animals. Furthermore, the complicated vaccine production process, requiring biosafety level 3 facilities, could hamper vaccine availability. Future challenges include the development of improved, easier to produce Q fever vaccines.

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

[Active surveillance for scrapie in the Netherlands: effect of a breeding programme on the prevalence of scrapie in sheep (2002-2010)?]. / Acht jaar actieve scrapiesurveillance in Nederland: het effect van het fokprogramma op de prevalentie van scrapie bij het schaap (2002-2010).

The susceptibility of sheep to scrapie is modulated by the prion protein (PrP) genotype of the animal. An ambitious voluntary scrapie control programme was started in the Netherlands in 1998, based on selection of rams with theARR/ARR genotype for breeding. This programme was followed by an obligatory programme in 2004; the programme has been voluntary since 2007. We monitored the prevalence of PrP genotype frequencies and the prevalence of scrapie in the Dutch sheep population between 2002 and June 2010. Results showed that selection for scrapie-resistant sheep resulted in an increase in the ARR allele frequency in the Dutch national flock from 37.5% in 2005 to 61.4% in 2009. Moreover, surveillance data showed that there was a significant decrease in the prevalence of scrapie a few years after the start of the obligatory breeding programme, from more than 0.2% in 2004 to 0.015% in 2009. This decrease is a consequence of the increased number of scrapie-resistant sheep in the Dutch sheep population. To date, the results and the models based on the data show that the selective breeding programme should be continued for several years in order to successfully eradicate scrapie. It will be important to monitor the PrP frequency and scrapie prevalence in the Dutch sheep population in the coming years.

Differentiation of ruminant transmissible spongiform encephalopathy isolate types, including bovine spongiform encephalopathy and CH1641 scrapie.

With increased awareness of the diversity of transmissible spongiform encephalopathy (TSE) strains in the ruminant population, comes an appreciation of the need for improved methods of differential diagnosis. Exposure to bovine spongiform encephalopathy (BSE) has been associated with the human TSE, variant Creutzfeldt-Jakob disease, emphasizing the necessity in distinguishing low-risk TSE types from BSE. TSE type discrimination in ruminants such as cattle, sheep, goats and deer, requires the application of several prion protein (PrP)-specific antibodies in parallel immunochemical tests on brain homogenates or tissue sections from infected animals. This study uses in a single incubation step, three PrP-specific antibodies and fluorescent Alexa dye-labelled anti-mouse Fabs on a Western blot. The usual amount of brain tissue needed is 0.5 mg. This multiplex application of antibodies directed towards three different PrP epitopes enabled differential diagnosis of all established main features of classical scrapie, BSE and Nor98-like scrapie in sheep and goats, as well as the currently known BSE types C, H and L in cattle. Moreover, due to an antibody-dependent dual PrP-banding pattern, for the first time CH1641 scrapie of sheep can be reliably discriminated from the other TSE isolate types in sheep.

Genetic variability of the PRNP gene in goat breeds from Northern and Southern Italy.

AIMS: To determine the variability of the prion protein gene (PRNP) in goats from Northern and Southern Italy. METHODS AND RESULTS: Genomic DNA isolated from goat blood was polymerase chain reaction (PCR)-amplified for the coding region of the PRNP gene and then sequenced. In total, 13 polymorphic sites were identified: G37V, T110P, G127S, M137I, I142M, I142T, H143R, R154H, P168Q, T194P, R211Q, Q222K and S240P (substitutions I142T and T194P are novel) giving rise to 14 haplotypes. Clear frequency differences between Northern and Southern breeds were found and confirmed by genetic distance analysis. CONCLUSIONS: Differences in allele distribution were found between Northern and Southern goats, in particular regarding the M142 and K222 alleles, possibly associated to scrapie resistance; philogeographical analysis supported the idea that Northern and Southern breeds may be considered as separate clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: In Italy only limited studies have been carried out on caprine PRNP genotype distribution; this study is important to fill this lack of information. Moreover the finding of significant differences among allele distributions in Northern and Southern goats, especially if involved in modulating resistance/susceptibility, need to be carefully considered for the feasibility of selection plans for resistance to scrapie.

Pathogenesis of bovine spongiform encephalopathy in sheep.

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrP(Sc)) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrP(Sc) was detected after 6 months in the tonsil and the ileal Peyer's patches. At 9 months postinfection, PrP(Sc) accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrP(Sc) accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrP(Sc) was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7-L1. At subsequent time points, PrP(Sc) was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrP(Sc) involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord.

Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks.

Susceptibility to scrapie in sheep is influenced by polymorphisms of the prion protein (PrP) gene, whereas no strong association between genetics and scrapie has yet been determined in goats due to the limited number of studies on these animals. In this case-control study on 177 goats from six Italian scrapie outbreaks, the association between PrP alleles and the occurrence of scrapie was studied. Three silent mutations and 11 PrP polymorphisms were identified, of which two polymorphisms (L133Q and M137I) and one silent mutation (T202T) have not been reported previously. Twelve alleles were determined by cloning. Statistical analysis suggested a possible protective role against scrapie for the glutamine to lysine mutation at codon 222.

Low frequency of the scrapie resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed.

Frequencies of polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene were studied in 1207 pure-bred and cross-bred Italian Biellese rams, a small ovine breed of about 65 000 head in Italy. Aside from the five most common alleles (VRQ, ARQ, ARR, AHQ and ARH), the rare ARK allele was also found, with the highest frequency reported so far in an ovine breed (2.5 %). ARK/--- genotypes had a total frequency of 4.9 %. The resistance-associated ARR allele was seen at a low frequency (8.3 %). Only 1.4 % of animals examined had a resistant ARR/ARR PrP genotype. Semi-resistant (ARR/ARQ, ARR/ARH and ARR/AHQ) PrP genotypes had a total frequency of 12.6 % and PrP genotypes that are associated with high scrapie susceptibility (e.g. VRQ/VRQ and ARQ/ARQ) had a total frequency of 81.1 %. Statistical analysis comparing PrP allele frequencies between pure-bred and cross-bred animals showed that the ARR allele occurred at a significantly lower frequency in pure-bred rams. Furthermore, comparison of PrP allele frequencies between pure-bred rams over 18 months of age and those below 18 months of age showed a significant decrease in the ARR allele in breeding rams over 18 months of age. Based on these results, breeding for scrapie resistance in the Biellese breed will have to take into account the low frequency of the ARR allele, which also seems to be subject to negative selection by farmers. Further investigation is required to understand whether the ARK allele is also associated with resistance to transmissible spongiform encephalopathies.

Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein.

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)-molecular size and glycosylation profile-in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.

Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk, and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes. Furthermore, the efficiency with which the protease-resistant PrP (PrP-res) of one species induces the in vitro conversion of the normal PrP (PrP-sen) of another species to the protease-resistant state correlates with the cross-species transmissibility of TSE agents. Here we show that the CWD-associated PrP-res (PrP(CWD)) of cervids readily induces the conversion of recombinant cervid PrP-sen molecules to the protease-resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrP(CWD)-induced conversions of human and bovine PrP-sen were much less efficient, and conversion of ovine PrP-sen was intermediate. These results demonstrate a barrier at the molecular level that should limit the susceptibility of these non-cervid species to CWD.